Transforming growth factor-.beta.1 (TGF-.beta.1) is a multifunctional regulator of cell growth and differentiation. It is capable of causing diverse effects such as inhibition of the growth of monkey kidney cells, (Tucker, R. F., G. D. Shipley, H. L. Moses & R. W. Holley (1984) Science 226:705-707) inhibition of growth of several human cancer cell lines, (Roberts, A. B., M. A. Anzano, L. M. Wakefiled, N. S. Roches, D. F. Stern & M. B. Sporn (1985) Proc. Natl. Acad. Sci. USA 82:119-123; Ranchalis, J. E., L. E. Gentry, Y. Agawa, S. M. Seyedin, J. McPherson, A. Purchio & D. R. Twardzik (1987) Biochem. Biophys. Res. Commun. 148:783-789) inhibition of mouse keratinocytes, (Coffey, R. J., N. J. Sipes, C. C. Bascum, R. Gravesdeal, C. Pennington, B. E. Weissman & H. L. Moses (1988) Cancer Res. 48:1596-1602; Reiss, M. & C. L. Dibble (1988 In Vitro Cell. Dev. Biol. 24:537-544) stimulation of growth of AKR-2B fibroblasts (Tucker, R. F., M. E. Olkenant, E. L. Branum & H. L. Moses (1988) Cancer Res. 43:1581-1586)and normal rat kidney fibroblasts, (Roberts, A. B., M. A. Anzano, L. C. Lamb, J. M. Smith & M. B. Sporn (1981) Proc. Natl. Acad. Sci. USA 78:5339-5343) stimulation of synthesis and secretion of fibronecfin and collagen, (Ignotz, R. A. & J. Massague (1986) J. Biol. Chem. 261:4337-4345; Centrella, M., T. L. McCarthy & E. Canalis (1987) J. Biol. Chem. 262:2869-2874) induction of cartilage-specific macromolecule production in muscle mesenchymal cells, (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. McPherson, A. Contin, N. R. Siegel, G. R. Galluppi & K. A. Piez (1986) J. Biol. Chem. 261:5693-5695) and growth inhibition of T and B lymphocytes. (Kehrl, J. H., L. M. Wakefiled, A. B. Roberts, S. Jakeoview, M. Alvarez-Mon, R. Derynck, M. B. Sporn & A. S. Fauci (1986) J. Exp. Med. 163:1037-1050; Kehrl, J. H., A. B. Roberts, L. M. Wakefield, S. Jakoview, M. B. Sporn & A. S. Fauci (1987) J. Immunol. 137:3855-3860; Kasid, A., G. I. Bell & E. P. Director (1988) J. Immunol. 141:690-698; Wahl, S. M., D. A. Hunt, H. L. Wong, S. Dougherty, N. McCartney-Francis, L. M. Wahl, L. Ellingsworth, J. A. Schmidt, G. Hall, A. B. Roberts & M. B. Sporn (1988) J. Immunol. 140:3026-3032)
Recent investigations have indicated that TGF-.beta.1 is a member of a family of closely related growth-modulating proteins including TGF-.beta.2, (Seyedin, S. M., P. R. Segarini, D. M. Rosen, A. Y. Thompson, H. Bentz & J. Graycar (1987) J. Biol. Chem. 262:1946-1949; Cheifetz, S., J. A. Weatherbee, M. L.-S. Tsang, J. K. Anderson, J. E. Mole, R. Lucas & J. Massague (1987) Cell 48:409-415; Ikeda, T., M. M. Lioubin & H. Marquardt (1987) Biochemistry 26:2406-2410) TGF-.beta.1, (TenDijke, P., P. Hansen, K. Iwata, C. Pieler & J. G. Foulkes (1988) Proc. Natl. Acad. Sci. USA 85:4715-4719; Derynck, R., P. Lindquist, A. Lee, D. Wen, J. Tamm, J. L. Graycar, L Rhee, A. J. Mason, D. A. Miller, R. J. Coffey, H. L. Moses & E. Y. Chen (1988) EMBO J. 7:3737-3743; Jakowlew, S. B., P. J. Dillard, P. Kondaiah, M. B. Sporn & A. B. Roberts (1988) Mol. Endocrinology. 2:747-755) TGF-.beta.4, (Jakowlew, S. B., P. J. Dillard, M. B. Sporn & A. B. Roberts (1988) Mol. Endocrinology. 2:1186-1195) Mullerian inhibitory substance, (Cate, R. L., R. J. Mattaliano, C. Hession, R. Tizard, N. M. Faber, A. Cheung, E. G. Ninfa, A. Z. Frey, D. J. Dash, E. P. Chow, R. A. Fisher, J. M. Bertonis, G. Torres, B. P. Wallner, K. L. Ramachandran, R. C. Ragin, T. F. Manganaro, D. T. Maclaughlin & P. K, Donahoe (1986) Cell 45:685-698) and the inhibins. (Mason, A. J., J. S. Hayflick, N. Ling, F. Esch, N. Ueno, S.-Y. Ying, R. Guillemin, H. Niall & P. H. Seeburg (1985) Nature 318:659-663)
TGF-.beta.1 is a 24-kDa protein consisting of two identical disulfide-bonded 12 kD subunits. (Assoian, R. K., A. Komoriya, C. A. Meyers, D. M. Miller & M. B. Sporn (1983) J. Biol. Chem. 258:7155-7160; Frolik, C. A., L. L. Dart, C. A. Meyers, D. M. Miller & M. B. Sporn (1983) Proc. Natl. Acad. Sci. USA 80:3676-3680; Frolik, C. A., L. M. Wakefiled, D. M. Smith & M. B. Sporn (1984) J. Biol. Chem. 259:10995-11000) Analysis of cDNA clones coding for human, (Derynck, R., J. A. Jarrett, E. Y. Chem, D. H. Eaton, J. R. Bell, R. K. Assoian, A. B. Roberts, M. B. Sporn & D. V. Goeddel (1985) Nature 316:701-705) murine, (Derynck, R., J. A. Jarrett, E. Y. Chem, & D. V. Goeddel (1986) J. Biol. Chem. 261:4377-4379) and simian (Sharples, K., G. D. Plowman, T. M. Rose, D. R. Twardzik & A. F. Purchio (1987) DNA 6:239-244) TGF-.beta.1 indicates that this protein is synthesized as a larger 390 amino acid pre-pro-TGF-.beta.1 precursor; the carboxyl terminal 112 amino acid portion is then proteolyfically cleaved to yield the TGF-.beta.1 monomer.
The simian TGF-.beta.1 cDNA clone has been expressed to high levels in Chinese hamster ovary (CHO) cells. Analysis of the proteins secreted by these cells using site-specific antipeptide antibodies, peptide mapping, and protein sequencing revealed that both mature and precursor forms of TGF-.beta. were produced and were held together, in part, by a complex array of disulfide bonds. (Gentry, L. E., N. R. Webb, J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt & A. F. Purchio (1987) Mol. Cell Biol. 7:3418-3427; Gentry, L. E., M. N. Lioubin, A. F. Purchio & H. Marquardt (1988) Mol. Cell. Biol. 8:4162-4168) Upon purification away from the 24kD mature rTGF-.beta.1, the 90 to 110 kD precursor complex was found to consist of three species: pro-TGF.beta.1, the pro-region of the TGF-.beta.1 precursor, and mature TGF-.beta.1. (Gentry, L. E., N. R. Webb, J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt & A. F. Purchio (1987) Mol. Cell Biol. 7:3418-3427; Gentry, L. E., M. N. Lioubin, A. F. Purchio & H. Marquardt (1988) Mol. Cell. Biol. 8:4162-4168) Detection of optimal biological activity required acidification before analysis, indicating that rTGF-.beta.1 was secreted in a latent form.
The pro-region of the TGF-.beta.1 precursor was found to be glycosylated at three sites (Ash 82, Asn 136, and Asn 176) and the first two of these (Asn 82 and Ash 136) contain mannose-6-phosphate residues. (Brunner, A. M., L. E. Gentry, J. A. Cooper & A. F. Purchio (1988) Mol. Cell Biol. 8:2229-2232; Purchio, A. F., J. A. Cooper, A. M. Brunner, M. N. Lioubin, L. E. Gentry, K. S. Kovacina, R. A. Roth & H. Marquardt (1988) J. Biol. Chem. 263:14211-14215) In addition, the rTGF-.beta.1 precursor is capable of binding to the mannose-6-phosphate receptor and may imply a mechanism for delivery to lysomes where proteolytic processing can occur. (Kornfeld, S. (1986) J. Clin. Invest. 77: 1-6)
TGF-.beta.2 is also a 24-kD homodimer of identical disulfide-bonded 112 amino acid subunits (Marquardt, H., M. N. Lioubin & T. Ikeda (1987) J. Biol. Chem. 262:12127-12131). Analysis of cDNA clones coding for human (Madisen, L., N. R. Webb, T. M. Rose, H. Marquardt, T. Ikeda, D. Twardzik, S. Seyedin & A. F. Purchio (1988) DNA 7:1-8; DeMartin, R., B. Plaendler, R. Hoefer-Warbinek, H. Gaugitsch, M. Wrann, H. Schlusener, J. M. Seifert, S. Bodmer, A. Fontana & E. Hoefer. EMBO J. 6:3673-3677) and simian (Hanks, S. K., R. Armour, J. H. Baldwin, F. Maldonado, J. Spiess & R. W. Holley (1988) Proc. Natl. Acad. Sci. USA 85:79-82) TGF-.beta.2 showed that it, too, is synthesized as a larger precursor protein. The mature regions of TGF-.beta.1 and TGF-.beta.2 show 70% homology, whereas 30% homology occurs in the pro-region of the precursor. In the case of simian and human TGF-.beta.2 precursor proteins differing by a 28 amino acid insertion in the pro-region; mRNA coding for these two proteins is thought to occur via differential splicing (Webb, N. R., L. Madisen, T. M. Rose & A. F. Purchio (1988) DNA 7:493-497).
The effects of TGF-.beta. are thought to be mediated by the binding to specific receptors present on the surface of most cells (Massague, J. et at. (1985) J. Biol. Chem. 260:2636-2645; Segarini, P. R. et at. (1989) Mol. Endocrino. 3:261-272; Tucker, R. F., et at. (1984) Proc. Natl. Acad. Sci. USA 81:6757-6761; Wakefield, L. M., et at. (1987) J. Cell Biol. 105:965-975). Chemical crosslinking of [.sup.125 I]-labeled TGF-.beta. to cell surface components has identified three receptor size classes having molecule weights of 53-70 kDA (type I receptor), 80-120 kDa (type II receptor) and 250-350 kDa (type III receptor). The type I and II receptors have been implicated in signal transducfion (Boyd, F. T. et at. (1989) J. Biol. Chem. 264:2272-2278; Laiho, M., et al. (1990) J. Biol. Chem. 265:18518-18524) while the type III receptor has been suggested to act as a storage protein (Segarini, P. R. et al. (1989) Mol. Endocrino. 3:261- 272). Little is known concerning signal transduction mechanisms which occur after receptor-ligand interaction.
The pleiotrophic effects of TGF-.beta. may be due to its ability to affect the transcription of other genes. TGF-.beta. has been shown to induce fos, myc and sis in AKR-2B cells (Leof, E. B., et al. (1986) Proc. Natl. Acad. Sci. USA 83:1453-1458):1453-1458) enhance expression of c-jun B in A549 cells (Pertovaara, L., et al. (1989) Molecular and Cellular Biology 9:1255-1264), increase the mRNA for matrix proteins (Penttinen, R. P., et al. (1988) Proc. Natl. Acad. Sci. USA 85:1105-1110), IL-6 (Elias, J. A., et al. (1991) J. Immunol. 146:3437-3446) and EGF-receptors (Thompson, K. L. et al. (1988) J. Biol. Chem. 263:19519-19528) and decrease expression of PDGF receptor .alpha. subunits (Battegay, E. J., et al. (1990) Cell 63:515-524). It alters the pattern of integrin expression in osteosarcoma cells (Heino, J., et al. (1989) J. Biol. Chem..264:21806-21813) and decreases the express of c-myc in keratinocytes (Coffey, R. J. et al. (1988b) Cancer Res. 48:1596-1602). TGF-.beta. induces expression of Il-1.beta., TNF-.alpha., PDGF and bFGF in human peripheral blood monocytes (McCartney-Francis, N., et at. (1991) DNA and Cell Biology 10:293-300).